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Aesthetic Plastic Surgery

, Volume 42, Issue 6, pp 1655–1663 | Cite as

Split-Face, Randomized, Placebo-Controlled, Double-Blind Study to Investigate Passive Versus Active Dermal Filler Administration

  • Mário Jorge Freire dos Santos
  • Ricardo Carvalho
  • Luis G. ArnautEmail author
Original Article Non-Surgical Aesthetic
  • 161 Downloads

Abstract

Background

QueryHyaluronic acid (HA) is a large polymer increasingly used as dermal filler. HA does not permeate through healthy skin and is administered using various injection techniques. As HA procedures become more popular, the number of complications in facial rejuvenation procedures is likely to increase. Alternative methods may be necessary to satisfy the increasing demand for HA procedures. High-frequency high-intensity ultrasound is a painless and noninvasive method to deliver large molecules to the skin that is expected to deliver HA with visible results.

Objective

Assess facial rejuvenation with HA delivered with high-frequency high-intensity ultrasound.

Methods

Fifteen women (mean age 55) willing to participate in a randomized, double-blind, face-split trial with HA and placebo formulations in different sides of the face, were subject to five treatment sessions with high-frequency high-intensity ultrasound. Photographs taken before the procedure and after the last procedure were evaluated by a panel of five experts, blind to which side was treated with the HA or with the placebo.

Results

The expert panel identified a noticeable facial rejuvenation in the HA side relative to the placebo with a very statistically significant difference between the two sides (p < 0.0001).

Conclusions

Administration of HA with high-frequency high-intensity ultrasound is safe and leads to unambiguous facial rejuvenation.

Level of Evidence I

This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Keywords

Hyaluronic acid Rejuvenation Wrinkles High-frequency ultrasound Penetration enhancement Piezoporation 

Introduction

Aging is a natural process, common to all living organisms. Some of its most visible signs are changes in skin appearance, namely wrinkles, sagging and dyspigmentation. The global increase in life expectancy, the decline in self-esteem with the loss of a youthful appearance and the pressure to prolong professional careers beyond 65 years of age, amplified the desire to rectify one of the most visible indicators of age: the skin.

The most dramatic histochemical change observed in senescent skin is the marked disappearance of epidermal hyaluronic acid (HA). HA is a high molecular weight, very anionic polysaccharide responsible for the water content of skin, where half the HA of the human body is present [1]. Although no major differences were observed between the total amounts of HA in young and naturally aged skin [2, 3], major differences in the distribution of HA in different skin extracts were found as a function of age, indicating a shift of HA toward lower dermal layers with aging [1, 2]. The amount of HA in normal stratum corneum is nearly half that of the epidermis, but whereas the stratum corneum has a smaller portion of high molecular weight HA (> 1000 kDa), it has a higher portion of low molecular weight HA (< 60 kDa) [4].

The natural ability of HA to attract and retain water molecules suggested its use to promote skin hydration and, consequently, skin firmness and turgor. The intradermal administration of HA was approved by the FDA in various formulations for the correction of facial wrinkles, nasolabial folds and lip augmentation, and HA rapidly became the most popular dermal filler. According to the American Society for Aesthetic Plastic Surgery, in 2016 nearly 2.5 million administrations of HA were performed in the USA.

Intradermal injections of water-based formulations containing HA are usually associated with pain and with transient and mild complications. The most common complications are injection site reactions (redness, swelling, bruising), hypersensitivity reactions, skin discoloration, vascular occlusion and opportunistic infections [5]. Injection-related pain is a major concern to patients and has been addressed with the addition of a local anesthetic such as lidocaine to the HA formulation [6]. Using a 100-mm visual analogue scale, a randomized, double-blind study reported a mean pain score of 63.5 mm immediately after injection, which was reduced to 25.3 when the formulation with lidocaine was employed [6]. Rejuvenation of the skin in the periorbital area is particularly challenging due to the presence of delicate anatomical structures. Injection in the superficial dermis of HA combined with glycerol improved the skin appearance but was associated with adverse events after the injection such as papules, erythema, edema, bruising and hematoma, in addition to pain during the injection [7]. It was reported that medium size papules lasted 5 days, edema (present in 80% of the treatments) lasted 6 days and hematoma (affecting 76% of patients) lasted 7 days [7].

It is widely recognized that the topical administration of drugs is only effective for molecules with molecular weights less than 600 Da and that hydrogen bonding exerts a retardant effect on diffusion through the stratum corneum [8, 9]. HA is a good example of a molecule that cannot be administered in topical formulations. Many physical methods have been developed to enhance the penetration of drugs in the skin, such as sonophoresis, electroporation, jet injectors, skin ablation or microneedles [10]. Of particular interest for aesthetic purposes are noninvasive methods that act fast, do not leave marks in the skin and allow the skin to recover rapidly after the end of the procedure. High-frequency high-intensity ultrasound pulses generated when short laser pulses are absorbed by materials that rapidly convert the laser energy into a thermoelastic expansion, were shown to increase the transepidermal water loss of healthy skin by a factor 2.5 and meet the criteria of interest for aesthetic applications [11]. Moreover, it was recently shown that such high-frequency ultrasound pulses enabled the delivery of 800 kDa fluorescein-labeled HA to the skin of minipigs [12]. This study reports the first clinical use of high-frequency high-intensity ultrasound pulses to deliver a molecule to human skin.

The difference between high-frequency sonophoresis and the ultrasound pulses used in this work needs to be emphasized. High-frequency sonophoresis employs low-intensity (0.2 W/cm2) ultrasound at 16 MHz or lower frequencies [13]. The high-frequency high-intensity ultrasound used in this work is a broadband ultrasound pulse generated in a photoacoustic (light-to-pressure conversion) process that yields frequency components above 100 MHz. Additionally, the peak compression pressures are high (Pmax > 10 atm) but very short lived (< 10 ns), and thermal effects are avoided by using a pulse repetition rate of 10 Hz. The photoacoustic process occurs when a pulse of a Q-switched Nd:YAG laser is absorbed by a material (“piezophotonic” material) that very efficiently converts the energy of the laser pulse into a thermoelastic expansion. Pmax and pulse repetition rate are controlled by the settings of the Nd:YAG laser. The high-frequency ultrasound pulses thus generated have pressure gradients in excess of 1 atm./ns that transiently perturb the structure of the stratum corneum and make it more permeable to large drugs [11]. The device to generate the ultrasound pulses used in this study was provided by LaserLeap Technologies SA (LL Dermal device).

We report a clinical study to assess the safety and efficacy of HA administration using high-frequency high-intensity ultrasound pulses generated by the LL Dermal device. The participants in the study accepted to be treated with two different protocols, one in the left and the other in the right side of the face. The ultrasound pulses were applied to both sides but only in one of them the gel formulation contained HA. The two formulations (with or without HA) looked identical. This face-split trial was double-blind since the participants and research clinicians were informed that one of the formulations did not contain HA, but did not know which one it was. In view of the decreased amount of low molecular weight HA in the stratum corneum of the aging skin, the active formulation contained non-reticulated 15 kDa HA to compensate for the loss of this natural component of the outer layers of the skin.

Materials and Methods

This split-face, double-blind, randomized, placebo-controlled trial evaluated a total of 15 participants (30 split faces) in two clinical centers. The study complied with the Declaration of Helsinki, the applicable national laws and regulations, the advice of the national regulatory agency and was approved by institutional review boards. Each participant signed and Informed Consent Form and a Consent for the Use of Photography before the study procedures were conducted. The research physicians performing the treatments, the participants of the study, the clinical centers where the study was conducted and the members of the evaluation panel did not receive any benefits form their involvement in this study.

Devices and substances employed

LaserLeap Technologies SA provided one LL Dermal device to each clinical center. This device has a Q-switched Nd:YAG laser operated at a pulse repetition rate of 10 Hz, a second harmonic generator and a diffusor to deliver a laser fluence of 50 mJ/cm2 at 532 nm. The device uses such laser pulses to generate the broadband ultrasound pulses via photoacoustic light-to-pressure transduction [11]. The ultrasound pulses have peak compressional pressures of 10 atm and frequencies extending to 100 MHz. All the laser pulse energy is converted in ultrasound, and no light exits the LL Dermal device. The laser fluence was checked before each clinical study and before the ex vivo investigation.

LaserLeap Technologies SA also provided two sets of identical 10 g flasks containing formulations #1 and #2. Formulation #1 contained 20% HA (HyActive, sodium hyaluronate > 93%, molecular weight 15 kDa, cosmetic quality class, produced by fermentation, provided by Contipro Biotech, Czech Republic), 20% H2O, 58% glycerol and 2% propylene glycol. Formulation #2 contained 2% ethanol, 86% H2O, 10% glycerol and 2% hydroxypropyl methylcellulose to match the viscosity as Formulation #1 and conceal the absence of HA. The compositions of these formulations were selected taking into consideration their safety, cosmetic convenience and also matched appearance and viscosity. The formulation number to apply in each side of the face was randomized and concealed in an envelope, which was opened by the research physician just before the beginning of the treatment. The research physician was not informed which one of the formulations (#1 or #2) contained HA and which one was the placebo. This was also an evaluator-blind study, as the panel of plastic surgeons and dermatologists did not know which side was HA and which side was the placebo when evaluating the photographs of the participants before and after the completion of the treatment.

Subject selection

Subjects were restricted to women with approximately symmetrical faces, between the ages of 30 and 70, willing to give informed consent and to adhere to requirements for study. The participants were informed that the study involved the dermal delivery of two cosmetic formulations, one of them containing hyaluronic acid. The participants were also informed that the intent of the procedure was to improve the hydration and smoothness of the skin and were specifically requested to abstain from the application of cosmetics or perfumes on the face in the days of the treatments, and to report every adverse event possibly related with the treatment (pain, erythema, itching, etc.). The participants were aware that the treatment protocols in the left and right sides of their faces were different and could lead to temporary asymmetries. They were also offered treatment at the end of the study to correct for asymmetry. The major exclusion criteria included: (1) dermatological disorders or wounds in the face, (2) laser or intense pulsed light treatment in the preceding 3 months, (3) use of injectable products in the face in the preceding 6 months, (4) pregnancy or breastfeeding, (5) known allergy to any component of the product under study, (6) use of anticoagulants, immunosuppressive, nonsteroidal anti-inflammatory drugs, (7) serious or autoimmune disease.

Study design

The study required 5 visits of each participant to the clinic. The first visit (Day 0) was dedicated to screening, informed consent and first treatment. The next 4 visits where dedicated to treatment. The visits were scheduled for days 3, 7, 11 and 15, but changes of ± 1 day were allowed for the convenience of the participants. Additional visits were offered to participants willing to correct facial asymmetries resulting from the treatment.

Patients were randomized to receive either a placebo or a HA formulation on each split-face. The left side of the face was identified with a violet hairpin and the right side with a rose hairpin, to avoid possible confusions. The left/right (violet/rose), placebo/HA formulation assignment was concealed in sealed envelopes. Before each first treatment, the research physician arbitrarily selected one envelope, wrote the name of the patient on the envelope, and opened it. The instructions in the envelope referred only to the assignment of formulation #1 to be applied in the rose (or violet) side, and formulation #2 in the other side, but did not disclose which formulation contained HA.

Each treatment lasted for 60 min, including 10 min for acclimatization and initial photography, 40 min of treatment and 10 min for cleanup and final photography. The research clinician took six photographs of each participant using the following views: profile left, ¾ left, front face, ¾ right, profile right and forehead. All efforts were made to ensure that the angles of the photographs were reproduced during the study. Flashes were not used and the illumination of the room selected for the photography was kept the same for the whole study. All photographs were taken in a relaxed position. The treatment consisted in the following procedure: (1) a 1 mm layer of the assigned formulation was spread in a first zone to be treated; (2) the LL Dermal device was gently pressed against the formulation and the skin and, using circular movements, exposed the skin to high-frequency high-intensity ultrasound for 10 min; (3) An extra 1 mm layer of the formulation was spread over the skin and allowed to stay in contact for 10 min while another zone was treated; (4) once the total exposure time (10 min of application + 10 min of contact) elapsed, the zone was cleaned with de-makeup tissues. The face was washed with neutral soap at the end of the treatment.

The 1-mm layer of formulation initially spread over the treatment area ensured a good acoustic coupling between the device generating the ultrasound pulses and the skin. The total exposure time of each treated area to HA or to the placebo was 20 min.

Evaluation

Once the study was completed, the non-clinical researcher organized the photographs in a slide show. Two slides using photographs from different angles for each subject, labeling the left side “violet” and the right side “rose,” were included in the slide show, i.e., a total of 30 slides were prepared. The criterion to select three of the five angles available was the best correspondence in expression, angle and illumination before and after the treatment. The slide show was made available to a panel of 5 plastic surgeons and dermatologists. The panel members were blind to the contents of the formulations applied and scored independently each set of photographs per participant. The instructions and scoring table is presented in Table 1. Once the scoring was completed, the placebo and HA sides of each participant were disclosed, average scores and standard deviations were calculated, and the statistical significance of the difference was evaluated. The evaluation of facial rejuvenation in placebo versus HA delivery with the LL Dermal device was based exclusively on the scores given by the independent panel of experts.
Table 1

Example of scoring table based on analysis of before and after photographs, evaluating wrinkles as a measure of facial rejuvenation with the criterion: 0—no change, 1—slight improvement, 2—improvement, 3—significant improvement

Examplea

Rose side

Violet side

X

2

0

aExample of classification to be used when the evaluator considers that the violet side does not present visible changes, probably because the placebo was used, whereas in the rose side some wrinkles were attenuated in one of the slides and in the other the skin looks smoother and brighter

Statistical Analysis

Average values of the scores given by the panel and standard error of the mean were obtained the whole population. The significance of the difference between the populations was analyzed using the Student’s t test for paired data.

Ex vivo permeation

The permeation of HA was also investigated ex vivo using fresh human skin from abdominoplasty. The formulation consisted of 10% HA (10 kDa, Contipro) with 1% of 20 kDa fluorescence-labeled HA (Creative PEGWorks). The formulation was applied over the skin, exposed to 10 min of ultrasounds generated by a LL Dermal device, and incubated for 10 additional minutes at 33 °C. A control without exposure to ultrasounds was performed under the same conditions. The skin was cleaned after the procedures and biopsies were collected for cryo-fixation. The frozen skin was cut into slices with thicknesses between 25 and 100 µm in a cryostat and kept refrigerated for confocal fluorescence microscopy.

Results

Figure 1 shows the confocal microscopy of three independent samples of fresh human skin subject to 10 min of high-frequency ultrasound followed by 10 additional minutes of contact of a formulation containing fluorescein-labeled 20 kDa HA. The fluorescence from fluorescein is clearly visible in the epidermis. Similar results were obtained in the permeation in minipig skin of 800-kDa hyaluronic acid linked to fluorescein [12]. The hyaluronic acid used in these studies is too large to permeate the skin passively. However, the high-frequency high-intensity ultrasound pulses generated by the LL Dermal device permeabilize the skin and significant amounts of HA can be found in the epidermis with less than 30 min of treatment. These results demonstrate that it is possible to deliver HA to the skin with noninvasive methods. The clinical study was designed to test the hypothesis that the amount of HA delivered in 20 min with such ultrasound pulses was enough to elicit a visible change in the skin.
Fig. 1

Confocal microscopy of hyaluronic acid labeled with fluorescein. Top—passive permeation for 10 min. Bottom—piezoporation for 10 min

Eighteen women were initially recruited for this study. One participant withdrew from the study for personal reasons. Two participants were excluded from the analysis, one because of the presence of visible and changing acne spots in the face and the other one because of an asymmetric face. One participant informed that she was being treated for an autoimmune disease. This participant was initially treated in a small area of the hand, did not show any adverse events and was included in the study. Fifteen participants completed the study and their photographs were included in the analysis. The average age of the participants was 55 years (min. 42, max. 66).

Only photographs taken before the treatment and after the last treatment session were included in the slide show. It consisted in two sets of photographs of each of the 15 participants, and each set portrayed different areas of the face. None of the photographs was edited by any means, except to crop the pictures to make then fit in each slide. As the colored hairpins were not visible in some of the cropped photographs, circular lines of rose or violet colors were included in the corresponding side of the face to avoid ambiguities in the classification of right/left sides of the face. Table 1 presents the instructions given in writing to the members of the panel. Each panel member worked independently at his/her own leisure. When the identity of the formulations and the randomization results were disclosed, it was possible to identify which side of the face was treated with the HA formulation (HA) and which side was treated with the placebo (Pl), for each participant. Table 2 presents the scores given by each member of the panel to each set of two slides representative of each participant. The photographs of a participant that the panel classified as “3—significant improvement” are presented in Fig. 2. Figure 3 presents a case where the average scoring of the panel members was “2—improvement,” and Fig. 4 presents a case of average scoring 1.6.
Table 2

Blind evaluation of the photographs before the treatments and after the last treatment by a panel of 5 plastic surgeons or dermatologists

Code

Key

Reviewer-1

Reviewer-2

Reviewer-3

Reviewer-4

Reviewer-5

R

V

HA

Pl

R

V

HA

Pl

R

V

HA

Pl

R

V

HA

Pl

R

V

HA

Pl

MO

Violet

0

1

1

0

0

1

1

0

0

1

1

0

0

1

1

0

1

2

2

1

MC

Violet

0

3

3

0

0

3

3

0

0

3

3

0

2

3

3

2

2

3

3

1

AP

Rose

2

1

2

1

1

0

1

0

3

0

3

0

2

1

2

1

2

2

2

2

LM

Rose

2

2

2

2

2

0

2

0

2

0

2

0

1

0

1

0

1

1

1

1

ID

Violet

2

3

3

2

0

2

2

0

0

3

3

0

0

1

1

0

1

2

2

1

MB

Rose

1

0

1

0

0

0

0

0

2

0

2

0

0

1

1

0

1

1

1

1

GB

Violet

1

2

2

1

0

2

2

0

0

1

1

0

0

1

1

0

1

2

2

1

MG

Rose

2

1

2

1

2

0

2

0

2

0

2

0

1

0

1

0

2

1

2

1

ML

Violet

1

1

1

1

0

1

1

0

0

1

1

0

0

0

0

0

1

1

1

1

CP

Violet

0

1

1

0

0

1

1

0

0

1

1

0

0

1

1

0

1

1

1

1

PC

Rose

2

1

2

1

0

0

0

0

1

0

1

0

1

0

1

0

1

1

1

1

ON

Violet

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

MP

Rose

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

1

1

1

PN

Violet

0

1

1

0

0

1

1

0

0

2

2

0

2

0

0

2

1

2

2

1

MA

Rose

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

0

0

0

0

AVG

   

1.53

0.60

  

1.20

0.07

  

1.60

0.00

  

1.00

0.33

  

1.40

0.93

R—rose, V—violet, HA—hyaluronic acid, Pl—placebo

Fig. 2

Example of a participant with an average score of 3.0 by the expert panel (active side is violet)

Fig. 3

Example of a participant with an average score of 2.0 by the expert panel (active side is rose)

Fig. 4

Example of a participant with an average score of 1.6 by the expert panel (active side is rose)

The scores given by the panel to the HA and Pl sides were averaged for each participant. The averaged values of HA and Pl of all participants were analyzed for statistically significant differences using the Student’s t test for paired data and p < 0.0001 was obtained. According to the blind-panel evaluation, the differences between the two sides are statistically very significant. The average and standard error of the mean for the placebo side was 0.39 ± 0.06, and increased to 1.35 ± 0.18 for the HA side. Figure 5 presents the results. A statistically significant difference between HA and Pl was also found in the subsets of evaluations consisting in the evaluation by each panel member. The panel found that there was a reduction in the extension of the wrinkles in the side of the face treated with HA in 93% of the participants. In only 1 participant did not respond to the treatment.
Fig. 5

Average scores given to each subject by the panel of experts

Discussion

It has been shown before that high-frequency high-intensity ultrasound pulses generated by materials with efficient light-to pressure transduction (“piezophotonic” materials), transiently permeabilize the skin and increase the dermal delivery of molecules of both low and high molecular weight [11, 12]. However, this is the first study attempting to show that the increased dermal delivery can have a visible effect on the skin. The molecule selected for this study was hyaluronic acid with a molecular weight of 15 kDa.

The primary objective of this study was to test the hypothesis that the increased dermal delivery of molecules with high-frequency high-intensity ultrasound observed in pre-clinical models could be translated into observable clinical effects. The split-face, randomized, double-blind, placebo-controlled study design and the evaluation of photographs taken before and after the end of the study by an independent panel of experts offered sufficiently objective evidence to verify this hypothesis.

It is important to emphasize that at least three alternatives could have been selected for the control side in this split-face study: (A) the procedure generates ultrasound pulses but the formulation does not contain HA, (B) the procedure does not generate ultrasound pulses but the formulation contains HA, (C) the procedure does not generate ultrasound pulses and the formulation does not contain HA. Option (A) was selected for this study because the absence of ultrasound pulses was more likely to be perceived by the clinical researchers and participants, than the absence of HA in the formulation. Admittedly, this placebo control uses high-frequency high-intensity ultrasound that may not be entirely neutral to the skin. Indeed, it has been reported that high-intensity pulsed ultrasound may improve facial wrinkles and skin laxity [14]. Hence, the evaluation of the procedure with HA + ultrasound may not have been evaluated against a totally neutral placebo but against an ultrasound treatment. This is the worse-case scenario for the evaluation of the permeabilization of the skin to HA, and was an additional reason to select this “placebo” for the control side in this study.

The plastic surgeons performing the study reported a high tolerability of HA administration with high-frequency high-intensity ultrasound pulses in the nearly 100 applications of this study. Only one transient and minor side effect was reported: redness after one treatment in one participant. This was assigned to insufficiently adherence to the treatment protocol: the participant was using makeup and did not clean it properly before the procedure. The redness resolved spontaneously and did not limit the participant to attend a social event in the evening of the same day of that treatment. There was no downtime and no pain with this procedure to administer HA.

The evaluation of this study was exclusively based on the judgment of the panel members. Nevertheless, each participant was asked to identify the side of the face treated with hyaluronic acid, without showing the photographs. Seven of the eight (87.5%) participants with an average score larger than 1 assigned the treated side correctly. Several of these participants requested treatment to correct for the asymmetry of the face after the completion of the study.

A proper evaluation of the longevity of treatments using this noninvasive delivery of HA would require the use of additional HA formulations, namely formulations with long-chain and cross-linked HA. The longevity of the facial rejuvenation is likely to depend on the combination between the high-frequency high-intensity ultrasound pulses and the nature of the HA employed. The results of this study encourage the use of larger cohort to test such combinations in a subsequent study.

Conclusions

This study evaluated the effect of the administration of hyaluronic acid with high-frequency high-intensity ultrasound pulses in facial rejuvenation versus the use of the same ultrasound pulses and a placebo formulation. The protocol was designed to provide the highest level of evidence with randomization, participant-blind, investigator-blind and evaluator-blind procedures in a face-split study. The study showed that the administration of HA with high-frequency high-intensity ultrasound is noninvasive and painless, the recovery is immediate and the administration is very safe: it is almost impossible to over-do. The treatment is very easy to perform, with a fast learning curve and predictable results. It allows for the treatment of cases where the use of needles may not be recommended. However, it is difficult to quantify the amount administered and, with the current hand piece, the precision of site treated is not better than ± 5 mm. The independent panel of experts found an improvement in facial rejuvenation of the side of the face treated with HA in 93% of the participants. The difference between facial rejuvenation with HA and high-frequency high-intensity ultrasound pulses versus ultrasound pulses and placebo was very highly significant (p < 0.001). The longevity of the treatment was not assessed in this study.

Notes

Acknowledgements

The authors wish to thank Dr. Gonçalo Sá and Ângela Correia for assistance with the ex vivo studies. Funding was provided by Fundação para a Ciência e a Tecnologia (Grant No. 007630UID/QUI/00313/2013), H2020 Research Infrastructures (Grant No. LASERLAB EUROPE 654148).

Compliance with Ethical Standards

Conflict of interest

LGA holds intellectual property rights and equity related to the technology for active dermal filler administration. MJFS and RC declare that they have no conflicts of interest.

Ethical Standards

All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery 2018

Authors and Affiliations

  1. 1.Cirurgia PlásticaCentro Cirúrgico de CoimbraCoimbraPortugal
  2. 2.Unit of Plastic and Reconstructive Surgery and Burns UnitUniversity of Coimbra HospitalsCoimbraPortugal
  3. 3.Centro de Cirurgia Plástica e EstéticaSanfilCoimbraPortugal
  4. 4.Chemistry DepartmentUniversity of CoimbraCoimbraPortugal

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